JP2000510331A - Method for detecting prostate-specific antigen used in monitor observation and diagnosis of prostate cancer - Google Patents

Abstract

  (57) [Summary] Provided are primers specific to prostate-specific antigens and methods for monitoring and diagnosing prostate cancer using these primers. A kit for diagnosing prostate cancer having these primers is also provided.

Description

The present invention claims the benefit of US Provisional Patent Application No. 60 / 015,765, filed on April 16, 1996. It is. Background of the Invention Prostate cancer is the most common malignancy in adult men, except for skin cancer, and is an ever-growing health problem in the United States. In 1994, 38,000 deaths resulted from the disease, and it was estimated that prostate cancer was the second most common cause of death in the same population, after lung cancer. If the cancer was early diagnosed as still limited to the prostate, the chances of cure would be significantly higher. Thus, there is a great need for a sensitive method to detect organ-confine prostate cancer. In recent years, malignant cells have been detected using the polymerase chain reaction (PCR) and its modification, and various malignant tumors have been diagnosed and monitored. For example, reverse transcriptase PCR (RT-PCR) is a powerful technique that can be used to detect the presence of a particular mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR, mRNA species is first reverse transcribed into complementary DNA (cDNA) using reverse transcriptase, and the cDNA is then amplified by a standard PCR reaction. Thus, RT-PCR can reveal the presence of a single type of mRNA by amplification. Thus, if the mRNA is very specific for the cell that produces it, RT-PCR can be used to identify the presence of a particular type of cell. Prostate-specific antigen (PSA), a 33 kDa protein, was initially thought to be produced by prostate epithelial cells and was thus used as a tumor marker to aid in the detection and monitoring of prostate cancer . Recent studies have revealed that PSA is not specific to prostate tissue. For example, Diamandis and Yu report PSA in 30% of female lung cancers, and more rarely in other tumors, in normal lung tissue and in biological fluids such as breast milk. Cl in. Chem. 1995, 41 (2): 177-179. PSA was also detected in endothelial tissue. RT-PCR of mRNA encoding PSA has been used in identifying prostate cancer cells. For example, Deguchi et al. Detected PSA mRNA in regional lymph nodes of patients whose prostate cancer had spread to the lymphatic system. Cancer Res. 1993, 53: 5350-4. An RT-PCR method for detecting prostate cancer cells in the systemic circulation of patients with advanced prostate cancer has also been disclosed. Moreno et al., Cancer Res. 1992, 53: 6110-2; Jaakkkola et al., Clin. Ch em. 1995, 41 (2): 182-186. However, patients with early prostate cancer and / or fully differentiated or local disease were found to be negative for PSA mRNA by PCR. Therefore, it was concluded that RT-PCR for PSA mRNA was not a useful tool for diagnosing or monitoring prostate cancer. Diamandis, EP and Yu, H .; Clin. Chem. 1995, 41 (2): 177-179. The present invention provides a novel RT-PCR method using novel primers for monitoring and diagnosing prostate cancer patients. This method is designed to measure the presence of mRNA for PSA in the bloodstream, thus providing evidence that prostate cells are circulating in the blood and confirming the diagnosis of cancer. In addition, circulation of prostate cells indicates that the patient is at risk for a secondary tumor or bone metastasis anchor. If the sample taken after prostatectomy or radiation therapy is reactive, it indicates that the prostate cancer has spread and the risk of surgery / irradiation being ineffective. SUMMARY OF THE INVENTION It is an object of the present invention to provide primers for use in the RT-PCR method for detecting prostate specific antigen for monitoring and diagnosing prostate cancer patients. Another object of the present invention is to provide a method for monitoring and monitoring the progress of prostate cancer, comprising detecting a prostate-specific antigen in a patient by RT-PCR using these primers. It is. Another object of the present invention is to provide a kit comprising these primers for monitoring and diagnosing prostate cancer. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows human DNA (SEQ ID NOs: 1, 8, 9, and 10) for prostate specific antigen (PSA). Introns are omitted for brevity. Exons are shown in bold. Primers previously disclosed in the art are underlined. The primers of the present invention, namely, DR PSA-Up (SEQ ID NO: 2), DR PSA-Down (SEQ ID NO: 3) and DR12mer (SEQ ID NO: 4) are shown in uppercase and underlined italics. DETAILED DESCRIPTION OF THE INVENTION The expression of mRNA specific to certain tissues in peripheral blood is thought to indicate the circulation of cancer cells and the metastasis of tumors originating from these cells. Although detection of PSA mRNA in peripheral blood is associated with metastatic prostate cancer, PSA mRNA has also been detected in the blood of patients with clinically limited prostate cancer. Therefore, clinical methods of staging prostate cancer often rank patients lower and underestimate the risk of metastasis. A novel method for detecting prostate-specific antigen by reverse transcriptase polymerase chain reaction, which can be used to monitor prostate cancer in patients, has been developed. As primers used in the method of the present invention, DR PSA-Up: 5'-GTTGTCTTCCTCACCCTGTCCG-3 '(SEQ ID NO: 2), DR PSA-Down: 5'-TCCAGCACACAGCATGAACTTG-3' (SEQ ID NO: 3) and DR12mer: 5 ' -GAATCACCCGAG-3 '(SEQ ID NO: 4). The method of the present invention involves isolating and washing buffy coat cells, isolating total RNA from buffy coat cells, subjecting them to a reverse transcription (RT) reaction for PSA, PCR for PSA, and PCR on a DNA sequencer. Detecting the presence or absence of the PSA product. The method further includes subjecting the housekeeping gene beta-2-microglobulin (B2G: Israeli et al., Cancer Research 1994 54: 6306-6310) to an RT reaction and PCR to monitor the amount of isolated RNA. Consists of In this method, prostate cancer is identified using Vacutainer Cell Preparation Tubes (CPT) (Becton Dickinson Vacutainer Systems Cat. No. 362761, containing sodium citrate anticoagulant, 8 ml extract, San Jose, CA). Collect blood samples from suspected patients, then centrifuge these samples according to the manufacturer's instructions to separate the buffy coat from other blood components, then wash the buffy coat cells with PBS, Centrifuge to pellet, dissolve the pelleted cells in cell lysis buffer, homogenize cells through a Qiagen QIA shredder microspin column, then swipe through Qiagen RNeasy column to bind RNA. And other components are removed, and the RNA is eluted with DEPC-treated water. NA is used as a substrate in the RT reaction, the RT reaction primer for the PSA reaction is a DR12mer specific for the PSA target (SEQ ID NO: 4), and then an aliquot of the RT reaction is combined with the primer DRPSA Up (SEQ ID NO: 2). And DR PSA Down (SEQ ID NO: 3) in a PCR reaction for PSA.In a preferred embodiment comprising RT reaction and PCR for B2G and monitoring the properties of the isolated RNA, The primer was 5'-AGCTTTGAGTGC-3 '(SEQ ID NO: 5), and the primers for the PCR reaction were 5'-AGCAGAGAATGGA AAGTCAAA-3' (SEQ ID NO: 6) and 5'-TGT TGATGTTGGATAAGAGAAT-3 '(SEQ ID NO: 7). The size of the product from each patient's response was determined by ABI GE using an ABI 373 DNA analyzer. Compare to ESCAN software, this combination requires the determination of the presence of one or less LN CaP (PSA producing prostate cancer tissue culture) cells at high dilution per million white blood cells (WBC). The average 8 ml blood sample will contain approximately 40 million WBCs.ABI672GENESCAN software measures the size of each base in the product and identifies each product in a limited manner. The specificity of the method of the invention in detecting prostate cancer was determined. PSA levels in blood samples from normal patients and patients with benign prostatic hyperplasia (BPH), prostatitis and various stages of prostate cancer were measured. The results of these experiments are shown in Table 1 below. Normal donors include men and women under 35 years of age. BPH donors include patients who are not cancer but have been diagnosed with BPH. Prostate cancer symptoms were determined by the patient's advisor. As shown by these results, the method of the present invention is specific for the measurement of metastatic prostate cancer, particularly prostate cancer in advanced stages and prostate cancer with less than 5 years of steady state. By "advanced state" is meant a cancer in which metastases to bone or other sites have been identified, or in which the metastases have spread and / or increased since the last examination. Patients with a steady state of less than 5 years experience a relapse rate of approximately 66% before 5 years empirically. The results shown in Table 1 are consistent with this recurrence rate. In tests with the assays of the present invention, two out of six non-reactive patients in this group remain stable after 5 years, while four out of six reactive patients may relapse. Is expected to be high. Samples from patients with stage B cancer or non-metastatic organ-restricted cancer were non-reactive with the methods of the present invention. In addition, a stable patient means a patient who has metastasized in the past but has not progressed for 5 years or more and is considered to be cured, Met. Recent studies have shown that prostate cell circulation can also occur as a result of prostatectomy. 0efe1ein et al. Urol. 1996 155: 238-242. If they are cancer cells, the circulation of these cells caused by the surgery will cause metastasis. The method of the present invention can be used to monitor these operations before surgery as well as after surgery to confirm that the patient is non-reactive. The present invention also provides a kit for monitoring and monitoring the progression of prostate cancer, comprising a primer of DR PSA-Up (SEQ ID NO: 2), DR PSA-Down (SEQ ID NO: 3) and DR12mer (SEQ ID NO: 4). Is provided. In a preferred embodiment, these kits also have primers for RT and PCR detection of B2G, such as SEQ ID NOs: 5, 6, and 7. Furthermore, the kit of the present invention may have a tube for collecting a blood sample, which can separate a buffy coat such as a Bactiner cell preparation tube from other blood components. The kit of the present invention may also have a means for isolating RNA. For example, in one embodiment, the kit comprises a cell lysis buffer, a Qiagen QIA shredder microspin column (Cat. No. 79655) for homogenizing cells, and a Qiagen RNeasy column (Cat. No. 74106). The kit also includes beta-mercaptoetanol (BME), diethyl pyrocarbonate (DEPC), Superscript II RNase H-Reverse transcriptase (Gibco BRL, Gaithersburg). , MD)), ethanol (200 proof), 100 mM dNTP, AmpliTaq DNA polymerase (Perkin Elmer Cat. N808-0153), 6% (6% T, 5% C) denaturing acrylamide gel Gib co / BRL) 6% sequencing Solution (Gel-Mix6; Cat. No. 5543UA), 10% ammonium persulfate, Blue dextran (50 mg / ml) and denaturing loading buffer containing 200 ml of formamide and 20 ml of 100 mM EDTA, sequencer Gibco / BRL Ultrapure・ Gel-mix la Including 1X TBE (Sequencer Gibco / BRL UltraPure Gel-Mixrunning mate) for cats (Cat. No. 15546-013) and Brij35 (Sigma Chemical Co. St. Louis, MO, Cat. No. 430AG-6) The reagents may include, but are not limited to, reagents and controls can also be included in the kits of the invention, for example, Genescan ROX-2500 standard (Perkin Elmer / ABI). Catalog number 401100), RNA from ATCC cell line CRL-1435 PC-3 (human prostate adenocarcinoma) or HTB-22 MCF7 (human pleural effusion, pulmonary adenocarcinoma) and ATCC cell line CRL-1740 LN CaP ( The present invention is further described by way of the following non-limiting examples, including, but not limited to, RNA from human metastatic prostate adenocarcinoma. EXAMPLES Example 1 Preparation of Reagents In sterile disposable plasticware essentially free of RNase, in glassware sintered at 180 ° C. for at least 8 hours or in polypropylene plasticware rinsed with chloroform. , Prepare reagents. Solutions for the RNA and reverse transcription operations are prepared using RNase-free glassware, autoclaved water and sintering spatella-treated chemicals for RNA operations. The solution water is treated with 0.1% DEPC at 37 ° C for at least 12 hours, then heated to 100 ° C for 15 minutes or autoclaved on a liquid cycle at 15 pounds per square inch for 15 minutes. Phosphate-buffered saline (PBS) used in the method of the present invention was prepared by dissolving NaCl (8 g), KCI (0.2 g), Na ₂ HPO ₄ (1.44 g) and KH ₂ PO ₄ (0.24 g) in distilled water. (800 ml). The pH of the buffer is adjusted to 7.4 with HCl. Then, to adjust the volume to 1 liter with H ₂ O. Prior to use, the buffer is sterilized by autoclaving on a liquid cycle at 15 pounds per square inch for 20 minutes and stored at room temperature. Lysis buffer (BME) is prepared from 100 μl BME per lysis buffer RLT (10 ml) in QIAGEN RNeasy kit. The lysis buffer / BME obtained is in a stable state for one month. Wash buffer RPE is prepared by adding 4 volumes of 96-100% ethanol to its concentrate obtained from Qiagen. Prepare 70% ethanol by adding 700 ml of ethanol (200 proof) to DEPC-treated water (300 ml). The solution is stored at room temperature. Example 2: Leukocyte isolation procedure A blood sample is obtained in a Bactiner CPT tube containing sodium citrate. The sample is then centrifuged at 1500-1800 RCF (relative centrifugal force) for 30 minutes in a horizontal vibrating head rotor at room temperature (20-25 ° C.). After centrifugation, the buffy coat appears as a whitish layer just below the plasma layer. Aspirate about half of the plasma without disturbing the cell layer. The cell layer is then collected with a sterile pipette and transferred to a 15 ml conical centrifuge tube. Add PBS to the conical centrifuge tube to bring the volume to 15 ml. The cells are then mixed with PBS by plugging the tube and inverting the tube five times. The tubes are then centrifuged at 300 RCF for 15 minutes and the supernatant is aspirated without disturbing the cell pellet. PBS is again added to the cells, bringing the volume to 10 ml. Plug the tube and mix the cells and PBS by inverting the tube 5 times. The tubes are then centrifuged at 300 RCF for 10 minutes. Aspirate as much supernatant as possible without disturbing the cell pellet. Lysis buffer / BME (400 ml) is added to the pellet and the cells are suspended by gentle agitation. At this stage, the sample is frozen at -70 ° C or used to immediately isolate RNA. Example 3 RNA Isolation Procedure The cell / lysis buffer / BME suspension is pipetted directly into a QIAGEN QIA shredder column and centrifuged at sufficient speed for 1 minute in a microcentrifuge. If liquid remains on the column, the time of centrifugation can be extended. A volume (400 μl) of 70% ethanol is added to the homogeneous lysate and mixed. The sample is then added to the RNeasy spin column and centrifuged at 8000 × g (10000 rpm) for 15 seconds. Discard the flow-through. The washing buffer RW1 (700 μl) is then pipetted onto the spin column. The column is centrifuged again and the flow-through is discarded. After this wash, the spin column is placed in a new 2 ml collection tube. Pipette wash buffer RPE (500 μl) onto spin column and centrifuge as above. Discard the flow-through. Pipette a second aliquot of wash buffer RPE onto spin column and centrifuge at full speed for 2 minutes to dry RNeasy spin column. This 2 minute spin does not allow any remaining ethanol to carry over to elution. The spin column was then transferred to a new 1.5 ml collection tube, the RNA sample was eluted with 50 μl of DEPC-treated water, transferred directly to the spin column membrane with a pipette, followed by centrifugation at 8000 × g for 60 seconds. Attach. The RNA sample may be used immediately for the RT reaction, or stored at -70 ° C until further use. Example 4: Measurement of RNA concentration The RNA concentration of each sample is measured with a UV spectrophotometer. First, the base absorbance of 75 μl of distilled water is measured at 260, 280 and 320 nm. Then add 5 μl of the RNA sample and mix. The absorbance of the sample is measured at 260, 280 and 320 nm. The neat RNA concentration is calculated as follows: a) subtract A320 from A260 to obtain the absorbance by the RNA; b) multiply by the dilution factor (80μ / 5μl); and c) convert factor (0.04mg / ml) ). Example 5: Reverse transcription operation of sample 2 μl of 50 μM PSA RT primer (SEQ ID NO: 4) is pipetted into each PCR reaction tube. Pipet 2 μl of 50 μM B2G RT primer (SEQ ID NO: 5) into another PCR reaction tube. Tubes may be prepared prior to storage at -20 <0> C. An RT reaction mixture is prepared immediately before use in the following manner. The number of sample reactants (PSA and B2G) to be tested is first determined as a variable X. Then mix the following volumes of reagents: 4 (X + 1) μl of 5XFirst Strand Buffer; 2 (X + 1) μl of 0.1 M DTT; 2 (X + 1) μl of 20 mM dNTP solution; 1 (X + 1) μl of SuperScript 11 reverse transcriptase. The solution is then stirred to homogeneity. Prepare two tubes per RNA sample, one tube for each reaction of PSA and B2G. Add 10 μl of RNA sample to each reaction tube. All RT reaction tubes are then placed in a thermocycler programmed at 70 ° C. for 10 minutes. As soon as the program is finished, put the thermocycler in PAUSE and place each tube on ice. Then add 10 μl of the RT reaction mixture to each tube and test the following program: 25 ° C. for 5 minutes; 42 ° C. for 15 minutes; 97 ° C. for 15 seconds; followed by at least 4 ° C. for at least 5 minutes. Example 6: Sample amplification operation A PSA PCR reaction mixture is prepared by the following method. The number of PSA sample reactants to be tested is determined as variable X. Then mix the following volumes of reagents: 31. 95 (X + 1) μl water; 4 (X + 1) 10 × PCR Buffer II; (X + 1) μl 20 mM dNTP; 0.8 (X + 1) μl 25 mM MgCl ₂ ; 2 (X + 1) μl PSA primer (SEQ ID NO: 2) And 3) and 0.25 (X + 1) μl of Taq enzyme. A B2G PCR reaction mixture is prepared in the following manner. The number of B2G sample reactants to be tested is determined as variable X. The following volumes of the reagents are then mixed: 15 (X + 1) μl water; 4 (X + 1) 10 × PCR Buffer II; (X + 1) μl 20 mM dNTPs; 0.8 (X + 1) μl 25 mM MgCl ₂ ; 8 (X + 1) μl of B2G primer (SEQ ID NOs: 6 and 7) and 0.25 (X + 1) μl of Taq enzyme. Prepare two tubes per RNA sample, one tube for each reaction of PSA and B2G. Add 40 μl of the appropriate reaction mixture to each tube with a pet. Pipet 10 μl of each RNA sample or control RNA reverse transcription reactant into each tube. For the negative (reaction) control, use 10 ml of water. The tube is centrifuged for 10 seconds in an Eppendorf microcentrifuge and all liquid is mixed at the bottom of the tube. The tube is then placed in a Perkin Elmer 9600 Thermocycler and cycled as follows: cycle type temperature time number of cycles cycle step 95 ° C 15 seconds 30 58 ° C 15 seconds 72 ° C 45 seconds hold 72 ° C 3 minutes 1 hold 4 ° C. Without limitation 1 Example 7: Sample detection operation Two 6 cm well-to-lead plates (one notched, 1 One is flat). Each plate is rinsed on both sides with distilled water. Select only one side of each plate as the inner surface (gel surface) of that plate. The inner surface is then rinsed with methanol, followed by 0.5% Brij. Repeat rinsing with methanol until the plate has no streaks. Next, a spacer is placed on the edge of the flat plate in a direction parallel to the short side of the plate. The scored plate is placed on the flat plate with the inner surfaces of the plate facing each other. The plate is then erected on its bottom edge and the spacer lined up so that the level is at the bottom and the gel surface. Slowly lay down so that the flat plate is flat at the bottom and clamp with six connecting clips. Add 75 ml ammonium persulfate to 20 ml Gel-Mix 6 and mix gently with mixing. The solution is then poured between the sequencing plates. Insert the comb sample and polymerize for 1-2 hours. After polymerization is complete, remove the clamp and clean the outside of the plate with distilled water, taking care to remove any acrylamide from the outside surface of the plate. The plate is then inserted into a 373 sequencer and the plate checked according to the manufacturer's instructions to check for interference. Fill the top and bottom buffer reservoirs with 700 ml and 300 ml of 1 × TBE, respectively. Remove the combed sample from between the plates and wash the acrylamide and urea from the wells with 1 × TBE. Pre-run the gel at 28 watts for 5 minutes. Pipet 2 μl of the PCR reaction into a 0.2 ml thin-walled PCR reaction tube. Then, add denaturing sample buffer (2X; 2.5 ml) and 0.3 ml of Genescan ROX-2500 preparation. Place tubes in PE9600 Thermocyc1er and test at 94 ° C for 10 minutes. At the end of the program, set the thermal cycler to PAUSE and place each tube on ice. Then load 4 μl of the cooled sample on the gel. The ABI sequencer is set to run at 28 watts, 600 volts, 40 mA for 3.5 hours and the Genescan data collection is collected for 3 hours. The expected values for each primer set amplification are as follows: Marker size (bp) Observed range B2G 537 529-540 PSA 586 585-588

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Claims (1)

[Claims]   1. A method for diagnosing and monitoring prostate cancer in a patient, comprising: No. 2, SEQ ID NO: 3 and SEQ ID NO: 4 using reverse transcriptase-polymer Of prostate-specific antigen mRNA in patient-derived samples by enzyme chain reaction A method consisting of:   2. Prostate specific antigen mRNA of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 Primers for detecting   3. Prostate comprising primers of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 A kit for monitoring and diagnosing adenocarcinoma.